The human gastrointestinal tract is home to trillions of microbial organisms, collectively known as the gut microbiota, that play a critical function in maintaining human health by providing the host with nutrients, promoting maturation of the immune system and preventing colonization by pathogenic organisms. However, more recently it has been appreciated that certain members of the microbiota can actually exacerbate disease associated with enteric pathogens. Therefore, there is a critical need to investigate the effect of commensal species on the virulence of intestinal pathogens. Enterohemmorhagic E. coli (EHEC) is an important human food borne pathogen that causes 265,000 cases of bloody diarrhea year in the United States, which in severe cases can progress to hemolytic uremic syndrome or even death. EHEC encodes two principal virulence factors that contribute to disease: a type three secretion system (T3SS) encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE), and Shiga Toxin. Previous studies have demonstrated that microbiota-derived products can modulate expression of these important virulence factors and affect the severity of disease. The objective of this proposal is to determine the effect of human gut commensals on EHEC associated disease. Our hypothesis is that members of the microbiota affect EHEC disease by modulating expression and activity of, and directly processing virulence factors. We have previously demonstrated that Bacteroides- produced succinate enhances LEE expression, leading to more severe EHEC disease. We will investigate the effect of other common gut commensals on EHEC disease and use metabolomics to identify the metabolites that may be eliciting these effects. We have also previously demonstrated that the presence of a common gut commensal, Bacteroides thetaiotaomicron (B. theta) leads to enhanced destruction of the host mucus layer during a mouse model of EHEC infection. We will investigate the mechanism of this enhanced mucus degradation and define the role of commensal mucosal glycan degradation in this process. Finally, we have demonstrated that B. theta-produced proteases are capable of cleaving EspB, a critical component of the EHEC T3SS, into two smaller products. We will identify the B. theta proteases responsible for this cleavage and functionally characterize the resulting cleavage products. Successful completion of the proposed work will reveal novel mechanisms of EHEC virulence modulation by the human gut microbiota. Understanding more about the factors that affect disease progression will allow us to design more effective therapies for this important human pathogen.